Salicylic acid (“SA”) (2-hydroxy benzoic acid), a phenolic compound, has been studied for its medicinal use in humans for more than 200 years (Vlot et al., “Salicylic Acid, a Multifaceted Hormone to Combat Disease,” Annu. Rev. Phytopathol. 47:177-206 (2009)) and its role as a plant hormone in disease resistance, leaf senescence, flowering and thermogenesis have also more recently been investigated (Vlot et al., “Salicylic Acid, a Multifaceted Hormone to Combat Disease,” Annu. Rev. Phytopathol. 47:177-206 (2009) and Raskin, “Role of Salicylic Acid in Plants,” Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:439-463 (1992)). The roles of SA in plant defense and the hypersensitive response (a fast form of programmed cell death or PCD) have been intensively investigated (Vlot et al., “Salicylic Acid, a Multifaceted Hormone to Combat Disease,” Annu. Rev. Phytopathol. 47:177-206 (2009) and Raskin, “Role of Salicylic Acid in Plants,” Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:439-463 (1992)). Leaf senescence is a slow form of programmed cell death that allows plants to mobilize nutrients released from senescing cells to seeds, storage organs or actively growing tissues (Zhang et al., “An ABA-Regulated and Golgi-Localized Protein Phosphatase Controls Water Loss During Leaf Senescence in Arabidopsis,” Plant J. 69(4):667-678 (2012) and Gan & Amasino, “Making Sense of Senescence: Molecular Genetic Regulation and Manipulation of Leaf Senescence,” Plant Physiol 113(2):313-319 (1997)). Although the role of SA in leaf senescence and its underlying molecular mechanism have been less studied, there is some evidence that both disease defense and leaf senescence seem to share some components in SA signaling and regulation (Love et al., “Timing Is Everything: Regulatory Overlap in Plant Cell Death,” Trends Plant Sci. 13(11):589-595 (2008) and Rivas-San Vicente & Plasencia, “Salicylic Acid Beyond Defence: Its Role in Plant Growth and Development,” J. Exp. Bot. 62(10):3321-3338 (2011)).
Much research has been carried out on SA biosynthesis. There are two SA biosynthetic pathways in plants: the phenylalanine ammonia lyase (PAL) pathway and the isochorismate (IC) pathway; both pathways use the primary metabolite chorismate (Dempsey et al., “Salicylic Acid Biosynthesis and Metabolism,” Arabidopsis Book 9:e0156 (2011)). The chorismate-derived L-phenylalanine can be converted into SA via either benzoate intermediates or coumaric acid through a series of enzymatic reactions involving PAL, benzoic acid 2-hydroxylase (BA2H) and other uncharacterized enzymes (Leon et al., “Benzoic Acid 2-Hydroxylase, a Soluble Oxygenase From Tobacco, Catalyzes Salicylic Acid Biosynthesis,” Proc. Nat'l Acad. Sci. U.S.A. 92(22):10413-10417 (1995)). Chorismate can also be converted to SA via isochorismate in a two-step process involving isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). In Arabidopsis, two ICS enzymes, which convert chorismate to isochorimate, have been identified; the IC pathway contributes approximately 90% of the SA production induced by pathogens and UV light (Wildermuth et al., “Isochorismate Synthase Is Required to Synthesize Salicylic Acid for Plant Defence,” Nature 414(6863):562-565 (2001) and Garcion et al., “Characterization and Biological Function of the ISOCHORISMATE SYNTHASE2 Gene of Arabidopsis,” Plant Physiol. 147(3):1279-1287 (2008)).
In plants, SA may undergo biologically relevant chemical modifications such as glucosylation, methylation and amino acid (AA) conjugation (Dempsey et al., “Salicylic Acid Biosynthesis and Metabolism,” Arabidopsis Book 9:e0156 (2011)). SA has been shown to be converted to SA sugar conjugates SA O-β-glucoside (SAG) and salicyloyl glucose ester (SGE) by SA glucosyltransferases (SAGT) (Lim et al., “The Activity of Arabidopsis Glycosyltransferases Toward Salicylic Acid, 4-Hydroxybenzoic Acid, and Other Benzoates,” J. Biol. Chem. 277(1):586-592 (2002) and Dean & Delaney, “Metabolism of Salicylic Acid in Wild-Type, ugt74f1 and ugt74f2 Glucosyltransferase Mutants of Arabidopsis thaliana,” Physiol. Plant 132(4):417-425 (2008)).
The SA glycosides are actively transported from the cytosol to the vacuole as an inactive storage form that can later be converted back to SA (Dean et al., “The Formation, Vacuolar Localization, and Tonoplast Transport of Salicylic Acid Glucose Conjugates in Tobacco Cell Suspension Cultures,” Planta 221(2):287-296 (2005)). Methylation inactivates SA but increases SA's membrane permeability and volatility, thus allows more effective long distance transport of this defense signal (Park et al., “Methyl Salicylate Is a Critical Mobile Signal for Plant Systemic Acquired Resistance,” Science 318(5847):113-116 (2007)). AA conjugation of SA at trace levels was found in infected Arabidopsis plants (Zhang et al., “Dual Regulation Role of GH3.5 in Salicylic Acid and Auxin Signaling During Arabidopsis-Pseudomonas syringae Interaction,” Plant Physiol. 145(2):450-464 (2007)). Recently, high levels of 2,3- and 2,5-dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA, respectively) sugar conjugates were detected in infected or aged Arabidopsis leaves, and they appeared to be the major inactive form of SA (Bartsch et al., “Accumulation of Isochorismate-Derived 2,3-Dihydroxybenzoic 3-O-beta-D-Xyloside in Arabidopsis Resistance to Pathogens and Ageing of Leaves,” J. Biol. Chem. 285(33):25654-25665 (2010)). SA in transgenic Arabidopsis plants expressing a bacterial salicylate hydroxylase (encoded by NahG) was shown to be converted to catechol; the NahG transgenic plants have thus been useful in plant defense and senescence studies involving SA (Friedrich et al., “Characterization of Tobacco Plants Expressing a Bacterial Salicylate Hydroxylase Gene,” Plant Mol. Biol. 29(5):959-968 (1995) and Yamamoto et al., “Salicylate Hydroxylase, a Monooxygenase Requiring Flavin Adenine Dinucleotide: I. Purification and General Properties,” J. Biol. Chem. 240(8):3408-3413 (1965)). However, the enzyme(s), presumably SA hydroxylases, responsible for the formation of 2,3- and 2,5-DHBA have yet to be identified in plants.
The present invention is directed to overcoming these and other deficiencies in the art.